Acetyl CoA carboxylase (ACC) is the rate-determining enzyme of fatty acid biosynthesis in plants and animals. ACC is a biotin containing enzyme which catalyzes the carboxylation of acetyl CoA to form malonyl CoA in a two-step reaction (Beaty & Lane, (1982). J. Biol. Chem. 257:924-929). The first step is the ATP-dependent carboxylation of biotin covalently linked to the enzyme. In the second step, a carboxyltransferase step, the carboxyl group is transferred to the substrate, acetyl CoA, to form malonyl CoA (FIG. 1). Citrate is a potent allosteric activator of ACC. Malonyl CoA is the C2 donor for de novo synthesis of long chain fatty acids.
In mammals, there are two subtypes of ACC, ACC1 and ACC2. ACC1 is mainly localized in lipogenic tissues such as adipose tissue and liver, where fatty acids are synthesized. ACC2 is found primarily in non-lipogenic tissues such as skeletal muscle and heart muscle, although some is also found in liver. Malonyl CoA allosterically inhibits carnitine palmitoyl transferase 1 (CPT1), which is a critical enzyme to transfer the long chain fatty acid into the mitochondria for β-oxidation. Because ACC2 is co-localized with CPT-1, the primary role of malonyl CoA that is synthesized by ACC2 has been suggested to regulate the rate of β-oxidation.
ACC is a potential target in metabolic diseases for the treatment of metabolic syndrome including obesity, insulin resistance and dyslipidemia. Increased rates of muscle fatty acid oxidation, a reduced fat content and a reduction in total body fat were observed in ACC-2 knock-out mice (Abu-Elheiga et al., (2001) Science 291:2613-2616; Abu-Elheiga et al., (2003) Proc. Natl. Acad. Sci. USA. 100:10207-10212). Harwood et al. reported that ACC inhibitors caused reduction in fatty acid synthesis, increase in fatty acid oxidation, and reduction of respiratory quotient in rats (Harwood et al., (2003) J. Biol. Chem. 278:37099-37111). Chronic dosing of these compounds resulted in the reduction of whole body fat mass and improvement of insulin sensitivity (Harwood et al., (2003) J. Biol. Chem. 278:37099-37111). These observations further validated the enzyme as a drug target.
Despite the importance of ACC in the discovery and development of ACC inhibitors as drugs for the treatment of metabolic diseases, technical difficulties associated with ACC have greatly hindered progress in this area. One of the technical challenges has been the lack of a convenient, robust, economical enzyme assay that would allow high-throughput screening (HTS) for ACC inhibitors in an efficient manner. A CO2-fixation assay is the most commonly used ACC assay (FIG. 2, scheme 1). In this assay, [14C]—NaHCO3, acetyl CoA, Mg-ATP, citrate and ACC are incubated at 37° C.; the reaction mixture is quenched with acid at the end of the reaction, followed by heating to remove bicarbonate as 14CO2. Scintillant is then added and the acid-stable malonyl CoA remaining in the vial is counted in a scintillation counter (Waite, M., and Wakil, S. J. (1962) J. Biol. Chem. 237:2750-2757, Tanabe et al., (1981) Methods Enzymol. 71 Pt C, 5-16). This is a multi-step radioactive assay, which is time consuming and labor-intensive. Further, this assay requires large amounts of radioactivity, and special laboratory design to trap 14CO2 liberated in the assay. Although the assay can be run using a 96-well microtiter plate format, at best it is a low-throughput assay format and not suitable for HTS.
The continuous ATP regeneration-coupled spectrophotometric assay is another assay format. In this type of assay, the ADP generated in the ACC enzyme reaction is converted to ATP by a pyruvate kinase/lactate dehydrogenase coupled enzyme system, and NADH disappearance is followed at 340 nm spectrophotometrically or fluorometrically (Tanabe et al., (1981) Methods Enzymol. 71 Pt C, 5-16; FIG. 2, scheme 2). The ATP-regeneration system is very sensitive to the presence of ATPases. Since ATPase is highly abundant in tissue or cell culture extract, a disadvantage of this assay is that it demands highly pure ACC protein (about 5 μg/assay) and is less sensitive. Additionally, colored compounds that have absorption at 340 nm wave length may give false negatives in the screening.
Yet another form of ACC assay is an ACC/FAS coupled assay (FIG. 2, scheme 3). In the ACC reaction, malonyl CoA is formed from acetyl CoA. Malonyl CoA can then be used as a substrate for FAS with NADPH as the cofactor. The reaction can be monitored by the rate of utilization of NADPH spectrophotometrically (Wakil et al., (1959) Biochim. Biophys. Acta 34:227-233). However, this ACC/FAS spectrophotometric coupled assay requires large amounts of pure ACC and FAS enzymes, making this assay potentially expensive, and very time and resource intensive. Further, the rate of NADPH utilization has to be measured kinetically making the assay less amenable for HTS.
The assays discussed above are not practical for HTS, which requires a robust, reliable homogeneous assay that requires only small amounts of the enzymes. To fill this long-felt need, the inventors developed a homogenous ACC assay in which ACC is coupled to FAS and the product, palmitic acid, is detected by scintillation proximity. This assay can be particularly powerful for HTS of potential ACC modulators. The present assay can be used routinely for compound evaluation and can be employed to establish structure activity relationships.